Article title: Expression of Astrocyte Elevated Gene-1 (AEG-1) in Human Meningiomas and Its Roles in Cell Proliferation and Survival
Journal name: Journal of Neuro-Oncology
Author names: Kyung-Jae Park, Mi Ok Yu, Na-Hyun Song, Doo-Sik Kong, Dong-Hyuk Park, Yang-SeokChae, Yong-Gu Chung, and Shin-Hyuk Kang
*Corresponding Author:
Shin-Hyuk Kang, M.D., Ph.D.
Department of Neurosurgery, College of Medicine, Korea University #126, 5-ga, Anam-Dong, Seongbuk-Gu, Seoul, 136-705, Korea
Tel: +82-2-920-5729, Fax: +82-2-929-0629,
E-mail:
Material and Methods
Tumor specimens and cell lines
Surgical tumor tissue samples, obtained from the Department of Neurosurgery, Korea University Anam Hospital and the Samsung Medical Center, were routinely fixed in formalin and embedded in paraffin. Some portions of each sample were stored at -70°Cand in liquid nitrogen. The samples were histologically graded according to the WHO classification system.The specimens included 30 benign (WHO grade I), 16 atypical (WHO grade II), 8 malignant (WHO grade III) meningiomas and 2 samples of morphologically normal dura. The use of the tissue specimens was approved by the Institutional Review Boards of Korea University Anam Hospital and the Samsung Medical Center. The human meningioma cell lines SF4068, SF4433, SF3061, and IOMM-Lee were a generous gift from Professor HeonYoo (National Cancer Center, Korea). The authenticity of cell lines used in this study has been proved by DNA (STR) profiling technique using AmplFLSTRidentifiler PCR Amplification kit (Applied Biosystems, Foster, CA) at Korean Cell Line Bank. The cells werecultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum in a 37°C incubatorsupplemented with 5% CO2.
Immunostaining
Sections of formalin-fixed paraffin-embedded specimens were deparaffinized with xylene, hydrated in graded alcohols, and rinsed with phosphate buffered saline (PBS). The tissue sections were soaked in citrate buffer at pH 6.0 and heated in a microwave oven for 8 min for epitope retrieval. After cooling the sections for 15 min on ice, endogenous peroxidase activity was blocked using 3% hydrogen peroxideand 10% fetal bovine serum for 30 min.The sections were then incubated with a rabbit anti-AEG-1 antibody at a dilution of 1:250 (Invitrogen, Carlsbad, CA, USA), and incubated with an anti-rabbitIgG. The bound antibodies were detected using the EnVision kit (DakoCytomation; Carpinteria, CA, USA) that uses diaminobenzidinechromogen as a substrate. The cell nucleus was counterstained using haematoxylin.the degree of AEG-1expression in the meningiomaspecimens were graded accordingto the percentage of positive cells and stainingintensity: no or weak expression, moderate expression, and strong expression.Scores for percentage of positive cells were assigned as follows: ≤10% of cells positive, 0; 11% to 25% of cells positive, 1; 26% to 50% of cells positive, 2; 51% to 75% of cells positive, 3; and >75% of cells positive, 4. Scores for staining intensity were assigned as follows: no staining, 0; light brown, 1; brown, 2; and dark brown, 3. Overall scores were obtained by multiplying the percentage score by the intensity score. Overall scores ≤3 were defined as no or weak expression, overall scores >3 but ≤6 were defined as moderate expression, and overall scores >6 were defined as strong expression.In the negativecontrols, PBS was used instead ofAEG-1 antibody. As a positive control, we used a glioblastoma tissue specimen. The scoring of the staining intensity was conducted in a single blind manner to preventa potential bias from any knowledge of the WHO grades.
Reverse Transcription PCR (RT-PCR)
Total RNA was extracted from the cell lines and tissues by using the TRIzol reagent (Invitrogen) and was reverse transcribed with the SuperScript kit (Invitrogen), according to the manufacturer’s instructions. A real-time RT-PCR was performed in the icycleriQ multicolor real-time PCR detection system (Bio-Rad, Richmond, CA, USA) by using iQ SYBR Green Supermix (Bio-Rad). The primers used for real time RT-PCR were: 5′-GCT TCT GCA GGA AAG AGG AA-3′and 5′-ACT CCA ACT CCT TCC CCA GT-3′for AEG-1 and 5′-GAG TCC ACT GGC GTC TTC AC-3′ and 5′-GTT CAC ACC CAT GAC GAA CA-3′ for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).AEG-1 was amplified by performing PCR for each sample in triplicate, and its amplification level was normalized using the amount of GAPDH from the same sample. Semi-quantitative RT-PCR was performed in the GeneAmp PCR system (Applied Biosystems, Foster city, CA, USA) by using the primers 5′-TGA AGG AGC CTG GGA AAC TA-3′and 5′-CAG CCT CCT CCA TTG ACA GT-3′for AEG-1 and 5′-CGA GAT CCC TCC AAA ATC AA-3′ and 5′-TGC TGT AGC CAA ATT CGT TG-3′ for human GAPDH. The amplified products were separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining. Quantitativeanalysis was done with the use of densitometry and standardized withreference to the GAPDH reading.
Western blotting
Whole-cell lysates were prepared from human meningioma cell lines or normal human dura. The lysateswere separated on 10% sodium dodecyl sulfate-polyacrylamide gel andtransferred onto Hybond enhanced chemiluminescence membranes (AmershamPharmacia Biotech, Arlington Heights, IL, USA). The membraneswere blocked using 5% nonfat milk in Tris-buffered saline containing0.05% Tween 20 at room temperature for 30 min and incubatedovernight at 4°C with anti-AEG-1at 1:500, (Invitrogen), anti-p-Akt,anti-Akt, and anti-Bcl-2 at 1:1000 (Cell signaling Technology, Irvine, CA, USA) and with anti-beta actin at 1:1000 (BioVision ResearchProducts, Mountain View, CA, USA) antibodies diluted in 1% bovineserum albumin. Anti-rabbit IgG (Amersham Life Sciences) and anti-mouse IgG (Jackson ImmunoResearch Laboratories,Inc., West grove, PA, USA) diluted 1:10,000 in 5%nonfat milk were used as secondary antibodies. The protein bands were detected using the enhancedchemiluminescence system (Amersham Life Sciences), according tothe manufacturer’s instructions.
Small Interfering RNA (siRNA) Transfection
Non-targeting and AEG-1siRNA were synthesized by Genolution Pharmaceuticals, Inc. (Seoul, South Korea) and annealed toform siRNA duplexes, according to the manufacturer’s instructions.Cells were seeded in 60-mm dishes at a density of 2 × 105 per dish and incubated for 24 h. The cells were transfectedwith 200nMsiRNAfor 6 h in Opti-MEM containing Lipofectamine™ 2000 (Invitrogen), and the knockdown effect of AEG-1siRNA was determined by real time RT-PCR and western blotting.
Cell proliferation
IOMM-LEE and SF3061 meningioma cell lines were seeded at a density of 1× 105 cells in each well of a 6-well plate and then transfected with AEG-1siRNA or non-targeting siRNA. The cells were then stained with trypan blue (Invitrogen) and counted using a hemocytometer at day 1, 2, and 3 after the transfection.Combined data from experiments done in triplicate were used for cell proliferation analysis.
Colony formation assay
At 48 h after transfectionwith AEG-1siRNAor non-targeting siRNA, 1 × 103meningioma cells were seeded in 60-mm dishes and incubated at 37°C under 5% CO2. After 14 days, the dishes were stained with 0.01% crystal violet overnight at room temperature. Colonies containing >50 cells were counted.Colony formation experiments were performed in triplicate.
Cell Cycle Analysis
At 48 h after non-targeting or AEG-1siRNA transfection, thecells were harvested and fixed with 70% ethanol. Fixed cells were washed with PBS and stained withpropidium iodide staining solution with 200 μg/mL RNase A in the dark and then analyzed in a flow cytometer(FACS Calibur; BD Biosciences, San Jose, CA, USA). Approximately 10,000 cells were counted from each sample. The percentage of cells in each phase of the cell cycle was determined using the CellQuest software (BD Biosciences).
Apoptosis Assay
The Annexin-V-fluorescein isothiocynate (FITC) Apoptosis Detection Kit (BD Biosciences) was used to detect apoptosis by flow cytometry, according to the manufacturer’s instructions. The cells were harvested and washed twice with ice-cold PBS, 48h after AEG-1or non-targeting siRNA transfection. The cells were then incubated with Annexin V-FITC and propidium iodide for 15 min in the dark at room temperature. Flow cytometric analysis was immediately performed in the FACSCaliburInstrument (BD Biosciences), and the percentage distributions of apoptotic cells were calculated using the CellQuest software(BD Biosciences).
Xenograft tumor growth in athymic nude mice
Non-targeting or AEG-1siRNA transfected IOMM-Leecells (1 × 106 cells in a volume of 0.1mL)were injected subcutaneously into the flanks of 3-week-old athymic nude mice. Tumor volumes were measured weekly with a caliper and calculated using the formula π/6 × larger diameter × (smaller diameter)2. All the experiments were performed with four mice in each group.